Effects and mechanism of shRNA－Piezo1 on apoptosis of nucleus pulposus cells under abnormal mechanical stretch stress

English Keywords:Nucleus pulposus cells  Piezo1 protein  shRNA interference  Ｍechanical stretch stress  Apotosis

English Abstract:

  【Abstract】 Objectives:　To investigate the effects and mechanism of shRNA-Piezo1 coding gene on apoptosis of nucleus pulposus cells induced by abnormal mechanical stretching stress.　Methods:　The nucleus pulposus cells from 8 cases of intervertebral disc herniation(Pfirrmann Ⅱ or Ⅲ) were isolated and cultured, and the second generation nucleus pulposus cells were used to construct the mechanical stretch stress model in vitro. The shRNA-Piezo1 interfering plasmid was constructed by shRNA interfering technique. The cells were divided into four groups according to the results of pretreatment: blank control group(the second generation of nucleus pulposus cells without mechanical stretching stress treatment), stretching stress group(the second generation of nucleus pulposus cells treated with mechanical stretching stress for 24 hours), shRNA negative control group(blank vector plasmid + 2nd generation nucleus pulposus cells with mechanical stretch stress treatment for 24 hours), shRNA interference group(shRNA-Piezo1 plasmid + 2nd generation nucleus pulposus cells with mechanical stretch stress treatment for 24 hours). Fluo 3-AM kit was used to detect the cytoplasmic Ca2+ level of the cells. Cell Meter kit was used to detect the mitochondrial membrane of the cells. Annexin　V-FITC kit was used to detect the apoptosis rate of the cells. Results:　(1)Collagen type Ⅱ and agrecan protein expression were positive in the cultured cells, which accorded with the characteristics of　nucleus pulposus cells.　(2)Lentiviral titer of 1×108TU/ml was the highest when the multiple transfection(MOI) of nucleus pulposus cells was 50. (3)The homo-3201 sequence was an effective sequence of shRNA-Piezo1. (4)There were significant differences in cytoplasmic Ca2+ content, mitochondrial membrane potential inversion ratio and apoptosis rate among the four groups(P<0.05); there was significant difference between the mechanical stress group and the blank control group(P<0.05), but there was no significant difference between the shRNA interference group and the shRNA negative control group(P>0.05); the shRNA interference group decreased significantly when compared with the stretch stress group and the shRNA negative control group(P<0.05), but there was no significant difference between the shRNA interference group and the blank control group(P>0.05). Conclusions: ShRNA Piezo1 can inhibit excessive apoptosis of nucleus pulposus cells under abnormal mechanical stretching stress by inhibiting cytoplasmic Ca2+ level and mitochondrial membrane potential inversion.